Geobacillus sp., a Thermophilic Soil
Bacterium Producing Volatile Antibiotics
Authors: Yuhao Ren, Gary Strobel, Joe Sears, and
Melina Park
This is a postprint of an article that originally appeared in Microbial Ecology on January 22,
2010. The final publication is available at Springer via
http://dx.doi.org/10.1007/s00248-009-9630-9.
Ren, Y., Strobel, G.A., Sears, J., and Park, M. 2010. Geobacillus sp. a thermophilic bacterium
producing volatile antibiotics. Microbial Ecology. 60: 130-136.
Made available through Montana State University’s ScholarWorks
scholarworks.montana.edu
Geobacillus sp. a Thermophilic Soil Bacterium
Producing Volatile Antibiotics
Yuhao Ren a, Gary Strobel,a* Joe Searsb, and Melina Parka
a
Department of Plant Sciences, Montana State University, Bozeman, Montana, USA,
59717
b
Center for Lab Services/RJ Lee Group, 2710 North 20th Ave., Pasco,Washington, USA
99301,
*Corresponding author: Tel. =1 (406) 994 5148; fax (406) 994 760
E-mail address: uplgs@montana.edu
Key Words – Soil bacterium, antibiotics, thermal vents, rDNA, thermophile
Running Head- Geobacillus VOCs
1
Abstract
Geobacillus, a bacterial genus, is represented by over 25 species of Gram positive
isolates from various man made and natural thermophilic areas around the world. An
isolate of this genus (M-7) has been acquired from a thermal area near Yellowstone
National Park, Montana, and partially characterized. The cells of this organism are
globose (ca. 0.5 μ dia.), and they are covered in a matrix-capsule which give rise to
elongate multi-celled bacilliform structures (ranging from 3-12 μ) as seen by light and
atomic force microscopy (AFM), respectively. The organism produces unique petal
shaped colonies (undulating margins) on nutrient agar and it has an optimum pH of
7.0 and an optimum temperature range of 55-65°C. The partial 16S rRNA sequence
of this organism has 97% similarity with Geobacillus stearothermophilus, one of its
closest relatives, genetically. However, uniquely among all members of this genus,
M-7 produces volatile organic substances (VOCs) that possess potent antibiotic
activities. Some of the more notable components of the VOCs are benzaldehyde;
acetic acid; butanal, 3-methyl-; butanoic acid, 2-methyl- ; butanoic acid; propanoic
acid, 2-methyl- and benzeneacetaldehyde. An exposure of test organisms such as
Aspergillus fumigatus, Botrytis cinerea, Verticillium dahliae, Geotrichum candidum
produced total inhibition of growth on a 48 hr exposure to M-7 cells ( ca.107) and
killing at a 72 hr exposure at higher bacterial cell concentrations. A synthetic mixture
of those available volatile compounds, at the ratios occurring in M-7, mimicked the
bioactivity of M-7. This work has interesting implications in the role of M-7 to other
organisms in its native environment.
2
Introduction
The Yellowstone National Park ecosystem in Wyoming, Idaho and Montana of
Northwestern United States is one of the most active thermal areas in the world. It holds
promise as a source of novel microorganisms that are adapted for survival under
extremely adverse conditions [3, 6, 9,11,12]. It is from this area that Thermus aquaticus
was originally isolated and, more recently, a plethora of other microbes [2]. Thus, in this
area we began a search for other thermophiles having unusual and biologically interesting
properties. One organism, answering the description of a bacilliform bacterium, appeared
in our selection process. The organism was genetically unique but seemed to be related to
the previously described bacterial genus-Geobacillus [1,7,10]. Furthermore, this
organism was demonstrated to produce volatile organic compounds (VOCs)[4,5].
Although bacteria, in general, have been described that make volatiles (VOCs) under
various conditions of fermentation, none of these are Geobacillus spp. [5]. Furthermore,
the VOCs of this organism possessed antimicrobial activity.
Since our isolate appeared to be related to the bacterial genus- Geobacillus, this group
belongs to Bacillus genetic group 5 which represents a phenotypically and
phylogenetically coherent group of thermophilic bacilli with high levels of partial 16S
rRNA sequence similarity (96.5–99.5%) [1,10]. Generally, these bacteria are rod-shaped
Gram-positive bacteria having growth temperature optima ranging from 35 to 78°C
[1,10]. Members of this genus grow aerobically or facultatively anaerobically and are
widely distributed in various naturally or artificially induced thermophilic areas such as
soils, hot springs, oilfields, hydrothermal vents, hay compost, hot water pipelines, heat
exchangers, waste treatment plants, burning coal refuse piles and bioremediation biopiles
[1,7]. Presently, this genus is represented by over 25 species [1,7]. Thus, this report
describes the isolation of Geobacillus sp. isolate M-7 and demonstrates that it, seemingly
uniquely, makes bioactive volatile compounds and shows that an artificial mixture of
many of the volatiles of this organism mimic the biological effects of the organism itself.
3
Materials and Methods
Collection of samples
A sample of soil was collected (5/6/08) from the soil in the northern area of the
Yellowstone ecosystem at: N 45° 05′ 461″; W 110° 46′ 573″. Samples were taken from
the inner layers of a small hole formed in the soil which was exuding white gases. The
soil was grey/green in appearance having a temperature at the time of collection at 55°C
and a pH of 6.7. The sample was designated M-7 and was immediately processed the
same day as sampling for its microbial content.
Enrichment and isolation
Enrichment and isolation of aerobic/thermophilic bacteria was carried out on nutrient
broth (NB) solution at 55 °C. Pure cultures were obtained by repeated transfers of serial
dilution cultures. The purity of the cultures was checked microscopically. A pure colony
arising from this sample was designated M-7 and kept on nutrient agar (NA) at 55°C.
Microscopy
Bacterial cells, from well developed bacterial suspensions, were observed after Gram
staining using a light microscopy. In order to have an idea of the surface structure of the
cells of M-7, atomic force microscopy (AFM) was carried out with a Nanoscope IIIa
Extended Multimode AFM from Veeco (Santa Barbara, CA) with a J-type scanner using
the methods of Suo et al. [15]. Briefly, a 2 week old, 100 µL turbid suspension of M-7 in
NB medium was brought to room temperature, placed on a freshly cleaved muscovite
mica disk, and kept at ambient conditions for 10 min before being rinsed with 100mM
sodium phosphate buffer and then dried with a stream of dry nitrogen. Silicon probes of
resonant frequency 200–400 kHz were used (Model TESP, Digital Instruments) and the
scan rate was 0.5 Hz.
Physiological characteristics
The bacterium (M-7) was grown in 100 broth of NB (pH 7.0) in an incubator under
varying temperatures. The growth was observed 35°C, 40°C, 45°C , 50°C , 55°C , 60°C,
4
65°C , 70°C , 75°C and 80°C, respectively. Likewise, the effect of pH on growth was
determined in the NB medium adjusted to the appropriate pH with HCl and NaOH
between 3.0 and 11.0 at 55 °C. Turbidity and plate counting methods were used to
determine bacterial growth characteristics.
Phylogenetic analysis
Bacterial genomic DNA was extracted from cell pellets (after cultivation on nutrient
broth for 3 weeks at 65 °C and it was obtained according to Stöhr et al [12]. The 16S
rRNA gene was amplified from isolated DNA using PCR. Each PCR mixture contained:
Standard Taq Reaction Buffer 5µl, Deoxynucleotide Solution Mix 200µM, Taq DNA
Polymerase 0.2units/µl (New England Biolab). Isolated DNA 5 µg/mL. Bac8f (5'AGAGTTTGATCCTGGCTCAG-3') 0.2 µM and Univ1392r (5'ACGGGCGGTGTGTAC-3') 0.2 µM. The thermal cycler protocol was 95°C for 4 min,
34 cycles of 95°C for 45 s, 54°C for 45 s, 72°C for 75 s, and a final 10 min extension at
72°C. Negative control reactions (no template) were routinely performed to ensure purity.
The PCR products were purified using the Purelink Quick Gel Extraction Kit (Invitrogen
corporation). Partial 16S rRNA gene sequences were compared to the GenBank database
by using BLAST. The size of 16S rRNA gene used for alignment was 839 nucleotides.
The partial DNA sequences were aligned and compared to each other using the
Molecular Evolutionary Genetics Analysis (MEGA) software version 3.3.14. A
phylogenetic tree was constructed using data from the BLAST search. The sequence was
deposited in the Genbank and was assigned the accession number FJ896054.
Comparison with related species and other microbes
A strain of Geobacillus stearothermophilus ATCC 7953, the closest genetic relative of
M-7, was purchased from the American Type Culture Collection (ATCC). It was
compared with M-7 relative to colony shape, and with images obtained by AFM as well
as its bioactivities. Other microbes used for testing were some common plant pathogens
that are usually used in bioassays in this laboratory. They represent a yeast-like fungus, a
Phycomycete, and several Fungi Imperfecti obtained from the Montana State University
living culture collection.
5
Analysis of M-7 gases
Gases were qualitatively analysed in the air space above cultures of M-7 grown for 21
days at 55 °C on 100 mL of NB in a sealed 250 mL brown glass bottle. Ultimately, the
bacterial suspension was transferred to a bottle having a Teflon-based septum for
analysis. First, a baked “Solid Phase Micro Extraction” syringe (Supelco) consisting of
50/30 divinylbenzene/carburen on polydimethylsiloxane on a stable flex fiber (gray) was
placed into the bottle containing the bacterial suspension and it was exposed to the vapor
phase for 45 min [13]. The syringe was then inserted into the splitless injection port of a
Hewlett Packard 6890 gas chromatograph (GC) containing a 30 m × 0.25 mm I.D. ZB
Wax capillary column with a film thickness of 0.50 mm. The column was temperature
programmed as follows: 30 °C for 2 min followed to 220 °C at 5°C/min. The carrier gas
was ultra high purity Helium (local distributor) and the initial column head pressure was
50 kPa. Prior to trapping the volatiles, the fiber was conditioned at 240°C for 20 min
under a flow of helium gas. A 30 sec injection time was used to introduce the sample
fiber into the GC. The gas chromatograph was interfaced to a Hewlett Packard 5973 mass
selective detector (mass spectrometer/MS) operating at unit resolution. The MS was
scanned at a rate of 2.5 scans per second over a mass range of 35-360 amu. Data
acquisition and data processing were performed on the Hewlett Packard ChemStation
software system. Initial identification of the compounds produced by M-7 was made
through library comparison using the NIST database. If possible, authentic compounds
were obtained from Sigma/Aldrich Chemical Co. and subjected to the identical analysis
as a means to confirm compound identity. The names of the organic compounds referred
to in this report follow that used in the NIST data base.
Bioassay test for volatile antimicrobials
A simple bioassay test system was devised that allowed only for an assessment of the
biological activity of the VOCs from the bacterium being tested. A glass rod supporting a
small plug of foam onto which was placed a piece of agar (potato dextrose agar-PDA)
containing hyphae of the test organism was placed into a sterile 10 mL screw capped vial.
6
At the base of the vial was placed the bacterial suspension to be tested (Fig 1). The
volatiles produced by the bacteria with antifungal activity were tested for their ability to
inhibit/kill test organisms. The NB medium without the M-7 culture was set up as a
control. The number of bacterial cells was increased in order to acquire killing of the test
organisms. Effectiveness of the M-7 in inhibiting and killing test microbes was recorded
as a function of bacterial concentration in the assay tube (Fig.1). The tests were repeated
at least three times with comparable results.
The antifungal activities of the artificial mixture and individual components of the M-7
VOCs were tested as previously outlined using some of the same test organisms [13].
Growth of the control assay cultures was ascertained relative to that of the assay cultures
in the presence of the bacterial culture and data recoded relative to the behavior of the
control. The IC50s (as μl/ml of air space above the Petri plate) of the artificial mixture of
the VOCs were determined using potato dextrose agar (PDA) plates containing
microcups having different volumes of the artificial VOC mixture [13]. The mixture was
prepared using the proportions (volumes) of individual compounds that appeared during
the GC/MS analysis of the M-7 volatiles. The experiments were repeated at least 3 times
and the data presented as averages with corresponding standard deviations of the means.
Results and Discussion
General biology and phylogenetics of Geobacillus sp. M-7
Geobacillus sp. M-7 grew optimally at pH 6.5-7.0, with a temperature optimum range of
55-65°C. The partial 16S rDNA sequence of M-7 indicated a 97% similarity to G.
stearothermophilus - ATCC 7953. The M-7 isolate produced acids and volatile organic
compounds (VOCs) with antibiotic activities in the NB. In contrast, G.
stearothermophilus did not produce VOCs having antibiotic activity. Aqueous glycerol
solutions (15%) have been placed in the MSU microbiological culture collection as
culture No.-2366 and held at -70°C. Soil from which the organism was originally
collected has also been kept at freezer conditions for re-isolation of the organism at the
MSU facility.
7
On the surface of nutrient agar, M-7 formed roundish, white colonies 1mm in diameter.
The colonies had irregular margins giving them a rough petal shaped appearance. This is
in contrast to G. stearothermophilus, one of the closest genetic relatives of this microbe,
whose colonies are white with smooth margins. The cells of M-7 are Gram positive and
are globose with a strong-solid matrix. They appear singly, or there may be several
arranged in a rod- like manner of varying lengths (Fig. 2). In contrast, other Geobacilli
usually appear as a definitive single rod- shaped cells or series of rod- shaped cells
aligned in a linear manner [7,10].
The individual cells of M-7 are globose with an irregular shaped matrix (Fig. 3). The
surface structure of G. evitos, as examined AFM, revealed an uneven surface but with
amorphous non- descript spreading margins represented by matrix material (Fig. 3). In
contrast, the surface G. stearothermophilus did not possess these features (data not
shown). It appears that M-7 has enough unique features for it to eventually be designated
as a novel species of Geobacillus.
Volatile organic compounds with antibiotic activity
Some bacterial species make VOCs with biological and economic potential [4,5]. Thus,
since cultures of M-7 possessed a distinctive odor, it seemed reasonable to ascertain if the
VOCs associated with this organism might have biological activity of the type that is so
distinctive of certain fungi in the fungal genus –Muscodor [13,14]. Thus, it was
necessary to devise a biological assay system in which the VOCs being emitted by the
bacterial culture could be exposed to the test microbe (Fig.1). Using this simple bioassay
technique it was possible to demonstrate that M-7 produced VOCs having relative broad
antifungal activities (Table 1). An exposure of test organisms such as Aspergillus
fumigatus, Botrytis cinerea, Verticillium dahliae, Geotrichum candidum produced total
inhibition of growth on a 48 hr exposure to bacterial cells ( in the range of 107). In fact,
both filamentous fungi and a yeast like- fungus, G candidum, were inhibited by the
bacterial VOCs (Table 1). The biological activities were measured as a function of the
concentration of the bacterial cells in the medium located at the assay tube. It seemed that
A. fumigatus, a human lung pathogen, was one of the more sensitive fungi to the bacterial
8
VOCs (Table 1). Also affected at higher concentrations of M-7cells were a series of plant
pathogenic fungi (Table 1). Although the test organisms were only 100% inhibited in
their growth under these conditions, when the concentration of bacterial cells was
increased by 0.5 to 1 order of magnitude then the death of each of the test organisms was
observed at a 72 hr exposure.
The chemical and biological nature of the VOCs of Geobacillus sp. M-7
A GC/MS analysis of the VOCs of M-7revealed the presence of a variety of
aldehydes, alcohols, acids, and other assorted compounds (Table 2). Benzaldehyde,
butanoic acid, 2-methyl- , and benzeneacetaldehyde were the major components (16.29,
13.9, and 6.56 in relative areas) of the VOCs of M-7 (Table 2). Those compounds with an
asterisk in the table were positively identified based on the acquisition of comparable
GC/MS data using authentic compounds. Interestingly, quinoline, 3-methyl-also
appeared as one of the VOCs as well as ethanone, 1-(2-aminophenyl)- and 2(3H)furanone, dihydro-4-methyl-. Those compounds (with asterisks) that could be secured
from commercial sources were tested individually and in combination for their biological
activity using the PDA plate bioassay system for artificial VOC mixtures as described by
Strobel et al., 2001[14].
Initially, an artificial mixture of the VOCs was tested at various levels using the microcup
assay technique to ascertain the IC50 values on each of the test organisms. The
composition of the test liquid contained each of the VOCs made by M-7 at the
concentration made by the fungus as ascertained by the relative peak area of GC/MS
analysis of the bacterial gases (Table 2). It appeared that Rhizoctonia solani and
Sclerotinia sclertiorum possessed the lowest IC50 values and thus seemed to be the most
sensitive to the artificial gas mixture. However, these organisms in addition to
Aspergillus fumigatus, Pythium ultimum, Trichodema viride, Ceratocystis ulmi and
Cercospora beticola required the smallest volume of the VOC mixture to cause 100 %
inhibition of fungal growth (Table 3). It appears that there was not a direct correlation
between the reaction of the test organisms to the M-7 VOCs and the artificial mixture of
VOCs other than Aspergillus fumigatus appeared to be one of the most sensitive
organisms to both of these VOC mixes (Tables 1 and 3). This might be related to the fact
9
that the entire set of bacterial VOCs were not used in the artificial mixture since they
were not available.
Interestingly, when the individual compounds were tested against all assay organisms,
consistently, the lowest IC50s of the various compounds tested were exhibited by
benzaldehyde followed by butanoic acid and then butanal, 3-methyl- (data not shown).
The other volatiles ranged from having little to no activity. Surprising, was the absence of
activity in quinoline, 3-methyl- since the quinolines form a base structure for such
important antibiotics as the actinomycins [15].
The ecology of Geobacillus sp.M-7
While M-7 could consistently be isolated from soils in the general area described in this
report its presence elsewhere in geothermal areas of the world is an open question. It
appears that it is a novel species of Geobacillus and additional efforts are needed to
establish this assumption. The VOCs of this organism are totally unlike those produced
by various species of Muscodor, thus it appears that there is no single effective
combination of volatile organic compounds that have antibiotic activity (13,14). The role
of these compounds in the biology of M-7 is even more uncertain. Their production,
under natural circumstances, is probably related to the kind and amount of substrates
available. Nevertheless, it is tempting to suggest that gas production by this organism in
nature plays some role in its successful ability to occupy certain niches by out-competing
other organisms.
Acknowledgements
The authors acknowledge the assistance of NSF grant CBET-0802666 in providing
support for this project. Ms. Suzan Strobel constructed the drawing represented in Fig. 1.
The Howard Hughes undergraduate research program at MSU supported the summer
stipend of Ms. Melina Park who worked on this project.
10
References
1. Abd Rahman RN, Leow TC, Salleh AB, Basri M (2007) Geobacillus zalihae sp. nov.,
a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia.
BMC Microbiol 10: 70-77.
2. Brock TD (1995) The road to Yellowstone-and beyond. Ann Rev Microbiol 49: 1-28
3. Johnson DB, Okibe N, Roberto FF (2003) Novel thermo-acidophilic bacteria isolated
from geothermal sites in Yellowstone National Park: physiological and phylogenetic
characteristics. Arch Microbiol 180:60-68
4. Kai M, Effmert U, Berg G, Piechulla B (2007) Volatiles of bacterial antagonists inhibit
mycelial growth of the plant pathogen Rhizotonia solani. Arch Microbiol 187 :351360.
5. Kai M, Haustein M, Molina F, Petri A, Schjolz B, Piechulla B (2009) Bacterial
volatiles and their action potential. Appl Microbiol Biotechnol 81: 1001-1012
6. Kozubal M, Macur RE, Korf S, Taylor WP, Ackerman GG, Nagy A, Inskeep WP
(2008) Isolation and distribution of a novel non-oxidizing crenarchaeon from acidic
geothermal springs in Yellowstone National Park. Appl Environ Microbiol 74: 942949
7.McMullan G, Christie JM, Rahman TJ, Banat IM, Ternan NG, Marchant R (2004)
Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus.
Biochem Soc Trans 32:214-7
11
8. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
9.Nakagawa S, Shtaih Z, Banta A, Beveridge TJ, Sako Y, Reysenbach AL (2005)
Sulfurihydrogenibium yellowstonense sp. nov., an extremely thermophilic, facultatively
heterotrophic, sulfur-oxidizing bacterium from Yellowstone National Park, and
emended descriptions of the genus Sulfurihydrogenibium, Sulfurihydrogenibium
subterraneum and Sulfurihydrogenibium azorense. Int J Syst Evol Microbiol 55 : 22632268.
10. Nazina TN, Tourova TP, et al (2001) Taxonomic study of aerobic thermophilic
bacilli: descriptions of Geobacillus subterraneus gen nov., sp., nov. and Geobacillus
uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus
stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus
kautophilus, Bacillus thermoglucosidasius and Bacillus therdenitrificans to
Geobacillus as the new combinations G. stearothermophilus, G thermocatenulatus,
G.thermoleovorans, G kaustophilus, G. thermoglucosidasius and G.
thermodenitrificans. Inter J Sys & Evol Micro 51: 433-446.
11.Sokolova TG, González JM, Kostrikina NA, Chernyh NA, Slepova TV, BonchOsmolovskaya EA, Robb FT (2004) Thermosinus carboxydivorans gen. nov., sp. nov.,
a new anaerobic, thermophilic, carbon-monoxide-oxidizing, hydrogenogenic
bacterium from a hot pool of Yellowstone National Park. Int J Syst Evol Microbiol
54:2353-2359.
12. Stöhr R, Waberski A, Liesack W, Völker H, Wehmeyer U, Thomm M (2001)
Hydrogenophilus hirschii sp. nov., a novel thermophilic hydrogen-oxidizing betaproteobacterium isolated from Yellowstone National Park. Int J Syst Evol Microbiol.
51:481-488.
12
13.Strobel, G.A., Dirksie, E., Sears, J., and Markworth, C. (2001) Volatile
antimicrobials from a novel endophytic fungus. Microbiol. 147: 2943-2950..
14.Strobel G. (2006) Harnessing endophytes for industrial microbiology. Curr Opin
Microbiol. 9: 240-4.
15. Waksman, S.A. (1967) The Actinomycetes. Ronald Press Co. New York.
13
Table 1. The biological activity of M-7 VOCs against a series of fungal pathogens. The
table shows the concentration of bacterial cells at the bottom of the assay tube required to
bring about a 100 % inhibition of the target test organism after a 48 hr exposure to the
atmosphere of the assay tube (Fig.1).
Fungal test organism
Aspergillus fumigatus
Geotrichum candidum
Sclerotinia sclerotiorum
Trichoderma viride
Botrytis cinerea
Fusarium solani
Pythium ultimum
Verticillium dahliae
Number of G. evitos cells per
assay tube
1×107
5×107
5×107
2.5×107
2.5×107
5×107
5×107
5×107
14
Table 2. A GC/MS analysis of the VOCs of M-7 after 21 days incubation
at 23 °C on the NB medium.
Retention time
(min)
3.772
3.843
8.441
12.224
12.346
13.215
13.602
14.342
14.450
14.716
14.839
18.590
19.956
20.842
Possible compound
Butanal, 2-methyl-*
Butanal, 3-methyl-*
2-Butanal, 2-methyl-*
Acetic acid*
2-Furancarboxaldehyde*
Benzaldehyde*
Propanoic acid, 2-methyl-*
Butanoic acid*
2(3H)-Furanone, dihydro-4methylBenzeneacetaldehyde*
Butanoic acid, 2-methyl-*
Phenol*
Quinoline, 3-methyl-*
Ethanone, 1-(2-aminophenyl)-
Relative
Area
1.42
4.85
0.75
4.17
0.79
16.29
3.03
3.25
1.41
MW
Daltons
88.15
86.13
88.15
60.05
96.09
106.12
89.11
88.11
100.23
6.56
13.9
0.41
0.33
0.34
120.15
102.13
94.11
143.19
135.17
*Indicates that an authentic standard compound possessed the relative same
retention time and mass spectrum as the compound originating with G. evitos.
15
Table 3. The effects of an artificial VOC mixture of M-7 on some target assay fungi
using the standard microcup assay on standard PDA plates. The assay was conducted as
per Strobel et al., 2001[13] with details in the Materials and Methods section. TheIC50s
were calculated from inhibition curves generated using varying amounts of the artificial
VOC mixture. The mixture was prepared using proportional amounts of authenticated
compounds as in Table 2. The test was run for 48 hr and then measurements made.
Test Organism
IC50s reported as
μL/mL of free air
space of the PDA
test assay plate
Rhizoctonia solani
Botrytis cinerea
Aspergillus fumigatus
Fusarium solani
Verticillium dahliae
Trichodema viride
Sclerotinia sclerotiorum*
Ceratocystis. ulmi
Geotrichum candidum
Cercospora beticola
Pythium ultimum
0.052±0.007
0.09±.002
0.067± .008
0.107 ±.0019
0.08±.003
0.104±.001
0.053±.015
0.072±.008
0.112±.003
0.091±.002
0.102±.005
16
Reported
Amount Needed to
Viability after Yield100%
48hrs of
Inhibition
Exposure to
μL
30 μL of
VOCs
Alive
10
Alive
20
Alive
10
Alive
30
Dead
20
Alive
10
Alive
10
Alive
10
Alive
20
Alive
10
Alive
10
Figure 1. The system used in doing VOC inhibition
assay tests using M-7 and various test organisms.
The bacterium was placed in the liquid medium at the
bottom of the tube. The test organism was placed on
the foam pad at the top of the tube suspended on a
small glass rod. The numbers reflect the tube’s
capacity in mL.
Figure 2. A Gram stained image of M-7
showing the rod –like character of the bacterium, but at
the same time nicely illustrating the globose nature of
the individual bacterial cells. The line is equivalent to 6μ.
17
Figure 3. An AFM image of Geobacillus sp. M-7.
Note the disorganized surface of the cells represented
by matrix material. The strands may represent flagellae.
18
19